Hemocyanins in spiders, XII. Dissociation and reassociation of Eurypelma hemocyanin.

The dissociation and reassembly of Eurypelma californicum hemocyanin was studied under various conditions of pH, ionic strength and protein, calcium and magnesium concentrations. The hemolymph concentrations of calcium and magnesium were determined to be ca. 4 and 0.9mM, respectively. Eurypelma hemocyanin does not dissociate upon dilution down to 0.04 mg/ml. At physiological pH, phosphate causes partial dissociation. Dissociation at alkaline pH requires two days for completion and is dependent on hemocyanin concentration. Reassociation, starting with the total dissociated subunits, yielded 37 S, 16 S and unreacted 5 S material. The yield of 37 S particles after one day was 75%, but rose slowly to 84% after two weeks. Raising the calcium concentration above 1mM resulted in an increase of 16 S particles (hexamers). High magnesium concentrations interfered with ordered reassembly. However, 37 S, native-like particles are readily formed in the absence of both calcium and magnesium. Reassociation is strongly favoured if ionic strength is increased. The significance of the 16 S reassociation product was studied by repeated dissociation and reassociation and analysis of subunit composition. It was found that the 37 S reassociate contained the whole set of subunits, their quantitative proportions being identical compared to native 37 S hemocyanin. In contrast, the 16 S material was lacking the dimeric subunit bc4 (= 4D). Evidently, this hexameric product arises by incorrect reassociation of monomeric subunits. P50 and nH values were recorded for the reassociated hemocyanins. The 37 S component had a higher affinity and lower cooperativity than native hemocyanin, possibly due to ageing of the subunits.